Generation of a Noncleavable Klotho Knockin Mouse to Dissect the Distinct Roles of Membrane-Bound vs. Soluble Klotho

Background: Klotho is a key regulator of mineral homeostasis that exists in membrane-bound (TM-KL) and soluble (sKL) isoforms. While both forms exert distinct biological effects, existing knockout models eliminate both, making it difficult to dissect their individual roles. Methods: We engineered a non-cleavable variant of Klotho (NCK) by mutating nine amino acids at the ADAM10/17 cleavage site near the transmembrane domain. These targeted changes prevent proteolytic shedding while preserving membrane localization and co-receptor function of Klotho. The NCK sequence was introduced into the murine endogenous Klotho locus using CRISPR-Cas9 genome editing to generate the NCK mouse. Results: In vitro, NCK is stably expressed, traffics to the plasma membrane, and retains full co-receptor activity with FGFR1 in response to FGF23. Unlike wild-type Klotho, NCK is highly resistant to proteolytic shedding from cells, even in the presence of overexpressed secretases. Generation of the NCK mouse was arduous due to multiple unintended mutations but we finally succeeded in a clean introduction into the mouse genomic Klotho locus, and its precise integration was confirmed by next-generation sequencing. We currently maintain a colony of F2 heterozygous NCK mice (from F1 × WT) which have normal appearance thus far, and intercrosses are underway to generate homozygous NCK animals for phenotypic characterization. Conclusion: This novel knock-in model segregates TM-Klotho from sKl in vivo, potentially eliminating, or greatly reducing circulating sKL while preserving cell expression function and signaling. It offers a powerful tool to investigate the distinct physiological and pathological roles of Klotho in systems biology. Funding: Private Foundation Support